Method for measuring the binding capacity of serum proteins



A ril 2, 1968 B. T. EBERLE 3,376,114

METHOD FOR MEASURING THE BINDING CAPACITY OF SERUM PROTEINS OriginalFiled May 2, 1960 IN VENTOR BYRON 7f EBERLE United States Patent Ofi ie3,376,114 Patented Apr. 2, 1968 3,376,114 METHOD FOR MEASURING THEBINDING CAPACITY F SERUM PROTEINS Byron T. Eberle, Waukegan, Ill.,assignor to Abbott Laboratories, a corporation of Illinois Originalapplication May 2, 1960, Ser. No. 26,044, now Patent No. 3,206,602,dated Sept. 14, 1965. Divided and this application Aug. 4, 1965, Ser.No. 489,449 4 Claims. (Cl. 23-230) This is a division of applicationSer. No. 26,044, filed May 2, 1960, now Patent 3,206,602.

This invention relates to an apparatus for measuring the bindingcapacity of serum proteins with thyroid hormone substances and to amethod for performing said measurement. In particular, the apparatus andmethod involves the use of a thyroid hormone substance labeled withradioactive iodine, namely, radio-L-triiodothyronine.

The measurement of the binding capacity of serum proteins with thyroidhormone substances has many useful applications, among which is themeasurement of thyroid gland function. One of the early and stillpracticed methods is the determination of basal metabolic rate by thecumbersome and tedious procedure of measuring oxygen consumption by asubject in the resting state. The advent of radioactive isotopesprovided the art with a basically new approach to the evaluation ofthyroid function. In particular, radioactive iodine or I provided theart with a useful tool for measuring activity of the thyroid gland. Theiodine uptake test is based on recording the uptake of iodine by thethyroid gland with a suitable detecting device such as a Geiger tube. Adose of iodine is given to the subject and the Geiger tube is placedproximate the area of the thyroid gland. A disadvantage of the test isthat any exogenous iodine detracts from the reliability of the test.

Another method which utilizes I is the conversion ratio test which wasbased on the principle that iodine is converted in the thyroid gland toa thyroid hormone substance called thyr-oxine. When I was administeredto a subject, the thyroxine hormone subsequently released into the bloodstream contained the iodine isotope and this iodine isotope would labelthe thyroxine and its subsequent protein forms which occurred aftermetabolic destruction thyroxine. The disadvantages of this methodinclude larger administration of a radioactive substance and repeatedvisits by the subject to the laboratory.

The art recognized that an in vitro test would be highly desirable andpreferred over the in vivo testing which necessarily requiresadministering radioactive substances to the subject. A test of this typewas developed which utilized radio-L-triiodothyronine, hereinafterreferred to as T-3. Radioactive iodine can be incorporated in the T-3molecule and this resulting labeled substance can be gainfully employedin an in vitro test. T-3 is a thyroid hormone substance like thyroxine,and both thyroxine and T-3 are known to be bound by plasma proteins andby red blood cells. These substances are most strongly bound to theglobulin fraction of the plasma proteins and then, to a lesser degree,to the albumin fraction of the plasma proteins. The practitioners of theart establish that these foregoing thyroid hormone substances had agreater affinity for the plasma proteins than for the red blood cells.The practitioners also discovered that the thyroxine substance was morefirmly bound by either the plasma proteins or the red blood cells thanwas the T-3 hormone substance. The discovery of the foregoing elementsenabled the practitioners to develop an in vitro test which provided thestepsof adding radioactive T-3, also referred to herein as T 3-1 towhole blood, measuring the activity of the whole blood, separating thered blood cells, determining the hematocrit and then determining theradioactivity of the red blood cells. Considerable data were collectedto establish that a hyperthyroid subject would be characterized by arelatively large amount of T3-I bound to the red blood cells and,conversely, a hypothyroid subject would have little T3-I taken up by thered blood cells.

This foregoing in vitro test was an important advance, but the test isnoted for annoying disadvantages. Many of these disadvantages ariseeither directly or indirectly from the hemolysis of red blood cellswhich adversely affects the reliability of the tests. Carefulprecautions cannot eliminate the possibility of hemolysis which canoccur even when fresh blood is stored for short periods of time. Inaddition, the problem of hemolysis is heightened because a salinewashing step is incorporated for the in vitro tests. It is obvious thatany hemolysis will discredit the reliability of the tests because suchdestroyed red blood cells have taken up the T3-I The tests have theadditional disadvantage of requiring centrifuging to pack the red bloodcells and the subsequent washing of said blood cells with isotonicsaline. The number of additional steps require a longer time to practicethem and also increases likelihood of error while executing thetechnique. Addi tional disadvantages characterized the method because ofthe necessity of incubating whole blood with T3-I An incubation periodis necessary in order to establish the degree of T3-I uptake by the redblood cells; however, with the whole blood method, the art requiredincubation at body temperatures of about 37 C. This requires the use ofa constant temperature bath with shaker. This incubation must also beconducted in a stoppered flask because carbon dioxide affects the T3-Iuptake by the red blood cells. This precautionary step is necessarybecause of some unknown relationship between carbon dioxideconcentration in the red blood cells and uptake of T3-I by said redblood cells.

It has been stated herein how the uptake by the red blood cells in theprior art method indicates the condition of thyroid function. The arthas also recognized that such uptake is affected by other physiologicalstates such as pregnancy wherein such uptake is decreased from normallevels.

It is, therefore, an object of this invention to provide an apparatusand method for efficiently determining the binding capacity of serumproteins for thyroid hormone substances.

It is another object of this invention to provide an apparatus andmethod which utilizes iodine labeled L-triiodothyronine as a tool.

It is still another object of this invention to provide an apparatus andmethod for evaluating thyroid function by conducting an in vitro testwith blood plasma.

It is a still further object of this invention to provide an apparatusand method which employs a resin sponge in place of red blood cells forthe in vitro determination of serum protein binding with thyroid hormonesubstance.

In the accomplishment of the foregoing objects and other objects whichwill be apparent, it is now provided that a sponge having an anionexchange resin incorporated therein is combined with a containersuitable for seating in a scintillation counter. A sponge with an anionexchange resin present therein is placed in a vessel to which is addedblood serum and a tracer amount of T3-I The thyroxine in the bloodplasma is most readily taken up by the globulin and albumin fractionscontained therein and, thereafter, the thyroxine and T3-l not taken upby the plasma proteins are bound by the resin within the sponge. Thetotal radioactivity of both the resin sponge and the serum is determinedby placing the container vessel in a scintillation counter. After asufficient number of counts have been made, the serum is removed fromthe tube and the sponge is Washed with Water. The Washing is facilitatedby squeezing the sponge with a depressor rod during the washing.Following the washing steps, the radioactivity present in the sponge isdetermined in the scintillation counter. The uptake of the T3 1 by theresin sponge is expressed as a percent of the total radioactivity in thecombined serum and sponge resin.

The tracer amount of T3-I can be prepared from available sources byproper dilution. The amount of activity should be a workable amount fortracer purposes. This will depend on, among other factors, the volume ofblood serum used and the sensitivity of the recording and detectinginstruments. This requirement is readily understood and can be readilyprepared by the skilled workers in the art. A commercial source of T3-Iis supplied by Abbott Laboratories under the trade name of Triomet. Thissource contains T3-l dissolved in 50% propylene glycol and has aspecific activity of 20-50 millicuries per mg. of L-triiodothyronine anda concentration of 0.5-0.6 mg. T3/ml. For the purposes of the instantmethod, the source of T3-I is diluted with isotonic saline or distilledwater to a concentration of 120 10 meg/0.1 ml. of solution. To thecontainer holding the 1 ml. of serum is added 0.1 ml. of the dilutedT3-I solution. The foregoing amount of T3-I provides an adequate amountfor the subsequent method, which amount also has an activ' ity in therange of 0.010.5 microcurie.

The foregoing preparation is made from a recently prepared source ofT3-I Since I has a half-life of about eight days, it is obvious that theradioactivity will diminish to low levels which will requirecorrespondingly greater amounts of T-3 for the method. Consequently, thedecay factor should be considered with preparations that have not beenrecently prepared. A workable tracer amount of activity in the solutionwould be around 3x10 counts per minute per 0.1 ml. of solution. Thistracer amount of activity can be determined by diluting the activity ofthe stock solution with a volume of isotonic diluent determined from theequation:

;tc. ml. of stock solution X 0.133

X decay factor=ml. of sodium chloride solution The decay factor can bedetermined from the Wellknown formula:

where A activity to be determined, A =initial known activity, 9:2.7183,t=elapsed time (from A to A in same units as T /z. T /2 =half-life ofisotope. Reference may also be made to easily available decay charts forindividual isotopes.

The resin sponge employed in this test comprises a polyurethane foam ofintercommunicating cell type containing 0.5 to 160, and preferably aboutto 70, parts by weight of a strong base anion-exchange resin per 100parts by weight of polyurethane matrix. Such a urethane foam resin isprepared by incorporating the ion-exchange resin particles in a mixtureof a polyether or polyester and a polyisocyanate and then subjecting themixture to the usual conditions for producing foams of polyurethanetype. The polyester employed in such process may be an alkyd oroil-modified alkyd having a molecular weight from 300 to 8000 obtainedfrom a dicarboxylic acid, such as adipic, phthalic, maleic, or sebacicacid, and a polyhydric alcohol, such as glycerol, ethylene glycol,diethylene glycol, trimethylolethane, and trimethylolpropane. Instead ofthe polyesters, there may be used polyethers having molecular weights inthe range of 300 to 8000 and formed from glycols having from 2 to 10carbon atoms. Examples of the glycols include ethylene glycol, propyleneglycol, trimethylene glycol, hex amethylene glycol, octamethyleneglycol, and so on. There may also be used block copolymers of ethyleneoxide and propylene oxide of the formulas 2 4= )a( 3 6 )l5( 2 4 )cH(CZII40)y(O3H60)x (Cs u )x( 2 40)yH /N-CH2-CH2N H(C2H40) (C3HsO)x(C3HflO)X(CflH40)yH where the average values of the subscripts may be asfollows:

a=1 to 6, b=12 to 40, 0:1 to 6, x=7 to 19, and 3 :1 to 3.

The alkyds may also be modified with fatty acids or esters such ascastor oil and in place of the dicarboxylic acids mentioned above, theso-called dimers of linolenic or linoleic acid may be used. The alkydmay be formed in the usual manner by reacting a mixture of thepolyhydric alcohol, the polycarboxylic acid, and any modifying fattyacid or oil in a common reaction vessel at a temperature of to 150 C. orhigher.

The diisocyanate used may be one or more of the following: ethylenediisocyanate, trimethylene diisocyanate, tetramethylene diisocyanate,pentamethylene diisocyanate, propylene-1,2diisocyanate,butylene-1,2-diisocyanate, butylene-1,3-diisocyanate,butylene-2,3-diisocyanate, and butylene-1,3-diisothiocyanate; alkylidinediisocyanates such as ethylidene diisocyanate (CH CH(NCO) butylidenediisocyanate CH CH CH CH(NCO) cycloalkylene diisocyanates such ascyclopentylene-1,3-diisocyanate, cyclohexylene-1,2-diisocyanate,cyclohexylene-l,4-diisocyanate; aromatic diisocyanates such asmphenylene diisocyanate, p-phenylene diisocyanate, 1-methylphenylene-2,4-diisocyanate, naphthylene 1,4 diisocyanates,o,o'-toluene diisocyanate; aliphatic-aromatic diisocyanates, such asxylylene-1,4-diisocyanate, xylylene- 1,3-diisocyanate,4,4-diphenylenemethane diisocyanate, and 4,4-diphenylenepropanediisocyanate. Toluene diisocyanates of the 2,4 and 2,6 isomeric formsare preferably employed to obtain fast reaction, but such isocyanates asdiphenyl methane-4,4-diisocyanate and p-menthane-diisocyanate may beused for a slower reaction or a more vigorous catalyst is employed. Theproportion of diisocyanate may be from 6 to 85% by Weight based on theweight of the alkyd or polyether. Within this broad range, it ispreferred to employ from 25 to of the diisocyanate on the weight of thealkyd or polyether.

While it is not essential that a catalyst be present, a tertiary aminemay be employed to advantage where it is desired to shorten the time ofreaction. As much as 2 to 10% of such a tertiary amine may be employedbased on the weight of the diisocyanate. Examples of the tertiary aminesthat are thus useful include the N-alkylrnorpholines in which the alkylsubstituent has from 1 to 18 carbon atoms of which N-methyl morpholineis typical, triethylamine, triethanolamine, dimethylethanolamine, N,N-diethylcyclohexylamine, and 1,4 diazabicyclo[2.2.2]octane.

The polyurethanes may be obtained merely by mixing the alkyd orpolyether with the diisocyanate and water with or without the catalystat normal room temperature up to C. The timerequired to effect thereaction and complete it may vary from 15 seconds to several hoursdepending upon whether a catalyst is employed, the activity of thediisocyanate, and the temperature. The mixture of the several reactantswith or without a catalyst may be placed in a mold in which it is formedinto the desired shape during the completion of the reaction. Likewise,a mixture of the reactants may be extruded continuously and, for thispurpose, the several ingredients and the temperature may be controlledso as to allow adequate time for the passage of the mixture from thepoint of mixing into the extrusion channel before setting occurs.

In order to stabilize the foam, an emulsifier may be employed. Theamount of emulsifier may be from 0.5 to 5% on the weight of alkyd andany of the usual emulsifiers may be employed. Non-ionic emulsifiers arepreferred, such as the ethylene oxide condensates derived from vegetableoils, such as castor oils, from alcohols, organic acids, phenols, andhydroxyesters.

In addition to the emulsifier, there is incorporated from 1 to 5%of'water based on the weight of polyester or polyether in order todevelop the necessary gas for formation of the cellular mass. In makingthe cellular products, the various ingredients may be mixed in differentways, depending on the resin/polyisocyanate system involved.

When it is desirable to manufacture the cellular ionexchange products ofthe present invention from polyesters on conventional continuousproduction from machines, the ion-exchanger resin is first mixed intothe polyester or polyether. If the mix exhibits too high a viscosity forpumping on such machines (e.g., if viscosity is appreciably above 25,000centipoises when measured on a Brookfield viscometer), aviscosity-reducing aid may be added in the form of a plasticizer orother viscosity-reducing liquids compatible with the foam system. Thepolyester (or polyether)/ionexchange resin mixture (with any necessaryplasticizer such as tris(fichloroethyl)phosphate or tricresyl phosphate)is in turn mixed with the diisocyanate and an activator mixtureconsisting of water and catalyst, and where necessary, an emulsifier. Inorder to produce the desired ion-exchange products by simultaneousmixing, the polyester (or polyether)/ion-exchange resin mixture issupplied to the mixing head from one line in the machine, thediisocyanate from another, the activator from still another separateline.

The foregoing anion resin polyurethane foam sponges can assume multipleforms, but a convenient and preferred embodiment prescribes acylindrical plug which can be seated neatly in the bttom of a containeradapted for placement in the well crystal or on top of a probe crystalof conventional scintillation counters. The actual physical dimensionsof a cylindrical plug of resin sponge will, of course, be determined bythe volume of serum used and by the size of the container which, inturn, will depend on the size of the seating well in the particularwellscintillation counter available to the practitioner. It is desirablethat once a resin sponge is prepared, a plug of standard size beemployed with a standard volume of serum in establishing the appropriateranges of T3I uptake for normal and other subjects undergoing theparticular study. Various modifications, of course, can be made in thetype of resin and the content thereof in the polyurethane foam; also, inthe make up and characteristics of the sponge. Modifications can also bemade in the volumes of serum and amounts of tracer material therein.Such variations will not detract from the operability of the method, butto attain the greatest advantage from the practice of the method, aselected volume of serum and a selected size of a particularly preparedresin sponge should-be adapted as standards.

FIGURE 1 is an embodiment of the invention.

FIGURE 2 presents a modification of the container of FIGURE 1.

FIGURE 3 illustrates a sponge partially collapsed by a depressor rod.

An embodiment of the vessel and sponge plug is illustrated in FIGURE 1wherein the vessel tube is shown seated in the well 5 of thewell-scintillation counter schematically represented as 8. The essentialrelationships of the plug 3 and vessel 2 are shown in the drawing. Thisrelationship includes the proper seating of the container '2 has a flatbottom 6: This embodiment of the container is preferred in the practiceof the method because the resin sponge 3 rests directly on the flatportion 6 and thereby is closer to the sensitive area of thescintillation phosphors or crystal. This results in a better countingefficiency. This particular embodiment also has a further advantage inthat the resin sponge tends to remain in a fixed position within thecontainer tube. This will be better understood by considering thesubsequent step of squeezing the resin sponge with a depressor rodduring the washing steps. Following the recording of the count for boththe serum and the resin sponge, the serum is removed and the sponge iswashed with distilled Water to remove any T31 which has not been boundto the resin sponge. To assure an effective washing, the sponge issqueezed with the aid of a depressor rod to force out the water absorbedtherein. FIGURE 3 illustrates the resin sponge 3 in the vessel 2partially collapsed by the depressor rod 7.

This squeezing step could possibly result in displacing thecharacteristic cylindrical volume of the resin sponge by wedging saidsponge in the rounded bottom portion of the vessel. It will be obviousthat such displacement will alter the geometry of the resin spongewithin the well of the well-scintillation counter. This altered geometrymeans that various distances from respective points on the surface ofthe sponge to the scintillation crystal have been changed by alteringthe configuration of the resin sponge. The likelihood of thisundesirable occurrence is reduced with the flat-bottomed containerillustrated in FIGURE 2.

The resilient nature of the polyurethane foam sponge contributesmaterially to the success of the method because it allows the washingstep to be more eflicient. As described hereinbefore, the radioactivityof the sponge itself must be measured so that an accurate percentage ofthe total radioactivity in the sponge and serum can be calculated. Thepercentage figure will be more meaningful if the radioactivity withinthe sponge truly represents the activity of the TE'a-I bound to theresin sponge. Any radioactivity arising from T3-I bound to serumproteins or not bound to either the resin sponge or serum proteinsshould be eliminated from the radioactive count. This radioactivecomponent is best eliminated by the efficient washing and squeezing ofthe sponge to facilitate the Washing step. In practice, it has beenfound that three or four washing steps are sufficient in order to obtaina meaningful radioactive determination of the T3-I bound to the resinsponge. It is apparent that these few simple washing steps would notattain their desired function if it were not for the resilient characterof the polyurethane foam.

The following examples are presented to illustrate the operation of theapparatus in determining the percentage uptake of T3-I in the practiceof the method. It is obviously intended that such illustrations are notintended to represent exclusive embodiments or steps practiced in exactexecution.

Example I To 500 grams of a polyether glycol having a molecular weightof approximately 3500 and an hydroxyl number of 65 was added 300 gramsof a strong base quaternary ammonium anion-exchange resin in the form ofthe chloride salt prepared by the process disclosed in U.S. Patent2,591,573 and having an anion-exchange capacity of about 4milliequivalents per gram of dry resin. This mixture was pumped to amixing chamber on a foam machine. From a separate reservoir, 174 gramsof an r :20 weight ratio mixture of the 2,4 and 2,6 toluene diisocyanateisomers was pumped to the mixing chamber.

From a third reservoir, 13.5 grams of a 28.6% aqueous solution oftriethylenediamine was pumped into the 'x 18" x 8". The foam had adensity of 2.3 lbs. per cubic foot and an ion-exchange resinconcentration equivalent .to 29.5% of the total foam weight.

Example II A round-bottomed polyethylene container conforming to-theshape of a conventional test tube suitable for seating in a well of ascintillation counter is used to receive a polyurethane foam-anion resinsponge prepared by the process of Example I. The sponge is cylindricalin shape and has a diameter of 1.2 cm. and a length of 1.2 cm. Severalmilliliters of whole blood are removed from a subject and placed in aseparate test tube. The red blood cells are separated from the blood bycentrifugation and 1 ml. of serum is decanted into the polyethylenecontainer. To this serum is added a tracer amount (IO-120x10 meg/0.1m1.) of radioactive L-triiodothyronine or T3-I having an activity ofabout 0.2() .25 microcurie.

The combined serum, resin sponge and tracer amounts of T3-I are allowedto incubate at room temperature with periodic mixing for about twohours. Thereafter, the container is placed in the well of ascintillation counter so that the crystal, shaped like a well, surroundsthe resin sponge within the container. The counts are recorded andcorrection is made for background. The container is removed and theserum is decanted from the container after which distilled water iswashed into the container, and the sponge is depressed several times ina squeezing operation to assure good washing. The distilled water isemptied from the container and the washing step is completed with anadditional amount of distilled water. The procedure is repeated and athird washing step is performed. The container is then returned to thewell of the scintillation counter and the counts of the radioactivityresiding in the sponge are recorded with correction for background as inthe previous recording. The percent uptake by the resin sponge of T3-Iis determined by the following equation:

Residual activity Initial activity X 100 where the initial activity isthe radioactivity as determined by the counts of the serum and resinsponge and the residual activity is the radioactivity residing only onthe sponge after the foregoing washing steps. The percent figuresprovide an index of percent uptake of T3-I by the resin sponge.

' .Example III 0.001 or one out of one thousand.

Subjects I Number Average glrgtake With Controls 54 30. 512. 9 32 44.2:1:6. 0 14 25. 312. 6 11 21. 6

'8 Example IV A polyethylene vessel having annular side walls and a flatbottom as illustrated in FIGURE 2 of the drawings is used to accept 021ml. of a diluted solution of T3-I prepared as described in Example II.To the vessel is then added 1 ml. of serum followed by a resin spongeplug of the type and size described in'Example II. The combined vessel,resin sponge, serum and T3-I are allowed to incubate at room temperaturefor about two hours, and the radioactivity of the combined serum andsponge is determined as in Example I. The serum is removed and thesponge is washed with distilled water three times according to theprocedure of Example I; and the residual activity remaining within thesponge is recorded.

The method described in the foregoing examples essentially provides forthe steps of separating red blood cells from Whole blood and adding atracer amount of T3-I to the resulting serum. The combined serum andT3-I are incubated and placed in a vessel suitable for seating in thewell of a scintillation counter. To the vessel is preferably added aresin sponge plug having dimensions which will allow said plug to besurrounded by the phosphor or crystal of the scintillation counter. Thenext step provides that the total radioactivity or initial activity ofthe combined serum and sponge is counted and recorded. Thereafter, theserum is withdrawn and the sponge is washed a few times with distilledWater with the addition of a squeezing Operation on the sponge with adepressor such as a glass or wooden rod. The activity taken up by thesponge or the residual activity is then determined in the scintillationcounter. The ratio of residual activity to the initial activity isexpressed as a percentage figure to indicate uptake by the resin spongeof T3-I Description of the apparatus and method in the foregoingexamples provides that the T3-I is mixed, from an available source, withthe serum in the vessel. It is intended that other procedures may beused to introduce the T3-I into the vessel. One of the alternativeprocedures could provide adsorption of a layer of the radioactivematerial onto the interior side Walls of the vessel. This method ofadding the radioactive material to the vessel provides dissolving theTEX-I in a volatile solvent such as n-butyl alcohol, then adding thesolution to the vessel, allowing the volatile solvent to evaporate withthe aid of mild heat or a stream of air over the vessel opening. Thecomplete evaporation of the volatile solvent will leave a thin layer ofthe radioactive material adhering to the side walls of the vessel. Smallvolumes of aqueous solutions can also be used to wet the side walls; thesubsequent evaporation Will also leave a dry layer of the isotope. Theamount of activity to be incorporated on the vessel Wall is determinedby the same considerations which are instrumental in determining thetracer amount of activity in the foregoing examples. A workable activityrange is about .0l-0.5 microcurie. The foregoing manner of introducingthe radioactive material into the vessel can be understood and practicedmore easily by referring to US. 2,911,338 wherein the method ofadsorbing radioactive substances on the surface walls of capsules istaught.

Another way to introduce TZi-I into the vessel is by means of an inertcarrier such as paper, cotton and the like. The carrier can be withdrawnfrom the vessel after the radioactivity has been removed therefrom. Theinert carrier can be conveniently fashioned to conform to a long stripor be affixed to a swab stick or the like in order to facilitatehandling thereof. It is additionally provided that a tracer amount of T3-1 can be placed directly on the resin sponge by contacting the resinsponge with a solution of T3-I and retaining said isotope on the resinsponge after removal of the solvent.

In the immediately foregoing embodiments it is there- 'fore providedthat the vessel will already contain the tracer amount of radioactivesubstance and the practitioner need only add the serum to the resinsponge within the vessel to practice the method.

The novel apparatus in this invention results in advantages because ofthe co-action of the vessel and the resin sponge contained therein. Thevessel is formed in the shape of a test tube so that it can be seatedwithin the crystal well or in a holder on top of a probe crystal of ascintilalation counter. As described hereinbefore, a preferredembodiment prescribes that the vessel has a flat bottom rather than around bottom in order to secure the resulting advantages of geometry andproximity of the resin sponge to the phosphor; and the fixed position ofthe resin sponge within the vessel. The vessel can be constructed of anymaterial which is adapted for operation in a scintillation counter. Itis obvious that such material should not unduly adsorb the labeledmaterial thereon, thus hampering the subsequent washing step, nor shouldit unduly interfere with transmission of the radioactive rays to thesensitive crystal. Among such materials are glass and the thermoplasticmaterials made by injection molding such as polyethylene, polypropylene,polystyrene and the like.

The scintillation counters employed in the process comprise the detectorunit and the required ancillary units which consist of an extra hightension unit and an amplifying, discriminating and scaling unit. Theforegoing units which comprise the scintillation counter are well knownto the skilled members of the art. The scintillation counters areparticularly adapted for the instant method because of the well andbecause I emits medium energy gamma rays which the scintillation counteris well adapted to receive and to count. The art contains many standardand conventional texts and statements which describe or illustrate boththe principle and the operation of the scintillation counter. Among manyother publications, the practitioner may consult the text, IsotopicTracers, by Francis Mulligan and Wormall, University of London, TheAthlone Press, 1959, pp. 142 et. seq.

Others may practice the invention in any of the numerous ways which willbe suggested by this disclosure to one skilled in the art. All suchpractice of the invention is considered to be a part hereof provided itfalls within the scope of the appended claims.

I claim:

1. A method for measuring the binding capacity of serum proteins withthyroid hormone substances which comprises the steps of mixing a traceramount of L- triiodothyronine labeled with radioactive iodine With bloodserum, placing in intimate contact with such mixture a resin spongecomprising a polyurethane foam of intercommunicating cell typecontaining 0.5 to 160 parts by Weight of a strong base anion-exchangeresin per 100 parts by weight of polyurethane matix, incubating themixture and said resin sponge, measuring with suitable detecting meansthe initial radioactivity of the combined mixture and the resin sponge,removing the serum, washing the resin sponge with water, and measuringwith suitable detecting means the residual radioactivity in the sponge.

2. A method for measuring the binding capacity of serum proteins withthyroid hormone substances which comprises the steps of mixing a traceramount of L-triiodothyronine labeled with radioactive iodine with bloodserum, placing in intimate contact with such mixture a resin spongecomprising a polyurethane foam of intercornmunicating cell typecontaining 0.5 to 160 parts by weight of a strong base anion-exchangeresin per 100 parts by Weight of polyurethane matrix, incubating themixture and the resin sponge at room temperature, measuring withsuitable detecting means the initial radioactivity of the combinedmixture and said resin sponge, removing the serum, adding water to theresin sponge, squeezing the resin sponge in the water, removing thewater and measuring with suitable detecting means the residualradioactivity in the resin sponge.

3. A method for measuring the binding capacity of serum proteins withthyroid hormone substances which comprises the steps of mixing a traceramount of L-triiodothyronine labeled with radioactive iodine with bloodserum, placing in intimate contact with such mixture a resin spongecomprising a polyurethane foam of intercommunicating cell typecontaining 15 to parts by weight of a strong base anion-exchange resinper parts by weight of polyurethane matrix, incubating the mixture andthe resin sponge at room temperature for about two hours, measuring withsuitable detecting means the initial radioactivity of the combinedmixture and said resin sponge, removing the resin sponge from themixture, washing the resin sponge with water 3-4 times, squeezing theresin sponge in the water, removing the resin sponge from the water andmeasuring with suitable detecting means the residual radioactivity inthe resin sponge.

4. A method for measuring the binding capacity of serum proteins withthyroid hormone substances which comprises the steps of mixing 1012'01O- micrograms of L-triiodothyronine labeled with radioactive iodine andhaving an activity of 0.02-0.25 microcurie to about 1 ml. of bloodserum, placing in intimate contact with such mixture a resin spongecomprising a polyurethane foam of intercommunicating cell typecontaining about 30 parts by weight of a strong base quaternary ammoniumanionexchange resin per 100 parts by weight of polyurethane matrix,incubating the mixture and the resin sponge at room temperature forabout two hours, measuring with suitable detecting means the initialradioactivity of the combined mixture and said resin sponge, removingthe resin sponge from the serum, washing the resin sponge with water 34times, squeezing the sponge in water, removing the resin sponge from thewater, and measuring with suitable detecting means the residualradioactivity in the resin sponge.

References Cited Mitchell, Resin Uptake of Radiothyroxine in Sera FromNon-Pregnant Women, Journal of Clinical Endoctrinology and Metabolism,vol. 18, pp. 1437- MORRIS O. WOLK, Primary Examiner.

R. E. SERWIN, Assistant Examiner.

1. A METHOD FOR MEASURING THE BINDING CAPACITY OF SERUM PROTEINS WITHTHYROID HORMONE SUBSTANCES WHICH COMPRISES THE STEPS OF MIXING A TRACERAMOUNT OF LTRIIODOTHYRONINE LABELED WITH RADIOACTIVE IODINE WITH BLOODSERUM, PLACING IN INTIMATE CONTACE WITH SUCH MIXTURE A RESIN SPONGECOMPRISING A POLYURETHANE FOAM OF INTERCOMMUNICATING CELL TYPECONTAINING 0.5 TO 160 PARTS BY WEIGHT OF A STRONG BASE ANION-EXCHANGEREISN PER 100 PARTS BY WEIGHT F POLYURETHANE MATIX, INCUBATING THEMIXTURE AND SAID RESIN SPONGE, MEASURING WITH SUITABLE DETECTING MEANSTHE INITIAL RADIOACTIVITY OF THE COMBINED MIXTURE AND THE RESIN SPONGE,REMOVING THE SERUM, WASHING THE RESIN SPONGE WITH WATER, AND MEASURINGWITH SUITABLE DETECTING MEANS THE RESIDUAL RADIOACTIVITY IN THE SPONGE.